Results

Results

 

Successful Amplification of 16S gene in E. Coli

            Experiments were preformed to determine a set of conditions that produced optimal results from allele specific PCR DNA testing. The 16S gene of E. Coli DNA was successfully amplified using 3mL of the 8F and 529R primers at an annealing temperature of 46˚C. The targeted band size of 530 base pairs was seen as the brightest band in lane five (Figure 1).

Successful Amplification of Control Primers

            Multiple tests of PCR found an annealing temperature of 55˚C optimal for amplifying a segment of wild type DNA using control primers. Using preferred PCR cocktail ingredients obtained from results of successful E. Coli band amplifications (1mL Taq polymerase, 5mL PCR buffer, 1mL MgCl₂, 2mL DNTPs and water) and manipulating the volume of primers and DNA in the cocktail an optimal set of conditions were found. Bands at targeted length of 182 base pairs long were found in lane three (Figure 2). The conditions for lane three were 3mL wild type DNA template and 1mL forward and reverse primers, these were considered the optimal conditions for successful PCR amplification of our control primers.

Successful Amplification of Site Directed Primers

            Site Directed Mutagenesis was carried out to manipulate a DNA sequence carrying the A4V mutation site found in some cases of Amyotrophic Lateral Sclerosis. Annealing temperatures varied from 50˚C to 58 ˚C. Bands at the length 1648 were expected and observed, along with non-specific binding, at annealing temperature 58˚C (Figure 3). Normal PCR cocktail ingredients along with 3mL wild type DNA template and 1mL of forward and reverse primers were used in successful amplification of a band at length 1648 base pairs. The band at the targeted length in lane three was brighter than the band in lane two which suggests that 58˚C is a better fit for the annealing temperature of these primers.

Amplification of Mutant primer on Wild Type DNA

            PCR amplification of mutant primers against wild type DNA served as a control. Designed mutant primers were run with wild type DNA in lanes one, two and three at 50˚C and 58˚C, and as expected no bands were found after analysis using gel electrophoresis (Figure 4).

Genomic Prep

            Genomic prep following the step of the Qiagen Kit yielded concentration of 0.002mg/mL of DNA. This number was found by analyzing purified DNA in a spectrophotometer.

Living with ALS:       

            A psychophysical analysis of ourselves after undergoing a week with symptoms of ALS showed that overall our daily lives became more difficult as the symptoms got worse. The amount of arm lifts able to complete in a span of a minute decreased linearly as the week progressed (Figure 6). A R² value of 0.922735 shows a strong correlation between time and number of lifts per minute. A R² value of 0.8607 over the seven days was recorded from the Stanford Health Assessment Questionnaire (HAQ) correlating progression of experiment with Index scores, as the time went up so did the index scores (Figure 7). Finally Index scores from the Roland Morris Disability Index were plotted against time of the experiment. Again, it was found that there was a strong correlation between progression of the simulated experiment and higher index scores, this is shown by a R² value of 0.8602 (Figure 8). T-test were performed on each set of data, plotting weight vs. number of lifts or index scores. For each test p values were found to be less than 0.0001 which means they are significant.

 

 

Interpretation

 

Experiment Summary

           Amyotrophic lateral sclerosis (ALS) is an autosomal dominantly inherited disease that is fatal usually within three to five years of the onset of symptoms (Orrell, 1999).  ALS causes degeneration of the nerve cells in the brain and spinal cord that control voluntary muscle movement.  Mutations on the SOD1 gene have been discovered that correlates to ALS in patients. It is still unclear how this mutation exactly causes the disease. The most common mutation, A4V, is a single base pair mutation on the 162^nd base pair of the SOD1 gene.  This mutation changes the amino acid of that codon from alanine to valine.  Previous research has shown that PCR can be used to identify this mutation (Rosen et al, 1994).  The question we are addressing is whether or not PCR can be used as a diagnostic test for this specific ALS linked mutation and more specifically to compare previously designed primers used in similar research to newly constructed primers using the ideas of Hidenobu Yaku (Yaku et al, 2008).  We hypothesized that our designed primers would discriminate against the mutant and wild-type DNA template. Our primers would do this because of a single base pair mismatch toward the 3' end, which is a method that has been shown to successfully discriminate against DNA templates (Yaku et al, 2008).

           To further understand the sociological aspect of having ALS, our team of researchers felt what it is like to have the disease first hand.  We measured our disability after portraying the disease on ourselves by using a professional survey.  Our experiment put limits on our physical ability, much like a person with ALS would experience.  We hypothesized that our mental health and physical strength would progressively get worse as the symptoms of the disease progress (reference). 

Original Predictions

           Our experiment was done via the use of polymerase chain reaction (PCR) using two forward primers; one for the mutant DNA sequence and one for the wild type DNA sequence, along with a reverse primer.  The primers we used were created such that the DNA sequence that we tested against has two mismatched base pairs. The point of this is to limit the possibility of the mutant primer from annealing to the wild type DNA and vice versa, thus creating a false positive (Yaku et al, 2008). The mutation is on the 3’ end of the primer sequence with the Yaku mismatch being two base pairs away from the mutation, which occurs at the 162^nd base pair. The reason for the position of the mismatches is to maximize the probability that the primers bind to the correct DNA (Yaku et al, 2008). Moreover, the forward primer for the wild type DNA also has an intentional mismatch on the third base pair from the 3’ end, the same position as the Yaku mismatch on the mutant forward primer. The rationale for this is that the wild type primer would now have two mismatches with the mutated DNA and it is much more unlikely that it would anneal to the mutated DNA, but still probable that it binds to the wild type DNA. The forward primers will anneal to the genomic DNA template base pairs 142-163 of the SOD1 gene, while the reverse primer will anneal to the genomic DNA template base pairs 752-773 of the SOD1 gene, creating the difference of 631 base pairs (Hattori, 2000).  When deciding upon the annealing temperature to be used, we analyzed the effects of a temperature that was both too high and too low. A high annealing temperature would provide the primer with too much energy and this would lead to the primer not annealing to the DNA strand (Don et al, 1991). If the temperature is too low, then the primers could exhibit non-specific binding, meaning that the primers would not anneal to the right spots and other possible sections of the DNA could be replicated (Don et al, 1991).

Results and Ultimate Findings

            We ran multiple experimental trials in order to optimize PCR conditions. Each trial consisted of adjustments to DNA concentration, primer concentration, and annealing temperature. We successfully ran a positive control with primers designed by Rosen et al. The gel electrophoresis of PCR ran with these primers clearly show the band size of 182 bp (Figure 2). Another positive control was the amplification of the 16S rDNA gene of E. coli cells. The 530 bp size band appeared on an agarose gel after running gel electrophoresis (Figure 1). In order to gather mutant DNA for our tests, we were required to run primers designed for site-directed mutagenesis on the wild-type DNA. These primers successfully annealed and created a 1648 bp long strand of DNA that contains the mutation (Figure 3). These primers also amplified many other locations, resulting in some non-specific binding. The primers designed to test for the A4V mutation did not create the predicted band size of 630 bp in length. The primers designed to anneal to the mutant DNA instead amplified a 230 bp long band. The primers designed to anneal to wild type DNA also amplified at different locations with some non-specific binding occurring.

            The sociological experiment was run to simulate symptoms associated with ALS and find a correlation between having these symptoms and feeling disabled. Each experiment ran was consistent among all group members. There was a clear decrease in the physical limit and endurance of the arms as weight was increased (Figure 6). This resulted in an average R² value of 0.9227 among our team. The disability index at Stanford University's Health Assessment Questionnaire (HAQ) also consistently increased as weight was increased and mobility was decreased with an average R² value of 0.8607 (Figure 7). Similarly, the Roland-Morris Disability Questionnaire Index (RMQ) increased in a very similar fashion with an R² value of 0.8602 (Figure 8).

Interpretation of Results

            The successful amplification of the E. coli gene helped us control the concentration of PCR cocktail ingredients such as PCR buffer, MgCl₂, Taq polymerase, and dNTPs. It also helped us control temperatures for the denaturation and elongation steps of PCR. Amplification of the A4V mutation using primers from a published paper helped us control the DNA template as well. Success of the PCR using site-directed primers meant that the primers had elongated with the mutation along with it. This resulted in a strand of DNA larger than our intended design primers with the mutation. This allowed PCR with our designed primers to be used with the site-directed PCR cocktail serving as the DNA template. The design primers, however, did not anneal to the mutated DNA template. They also didn't anneal to the wild type DNA template. These results lend support against our hypothesis that our primer design could be used as a diagnostic test for the A4V mutation on the SOD1 gene. This means that the assay we designed cannot be used to diagnose ALS patients.

            These results seem to be evidence against the work of Yaku and Rosen, whose primer design methods were used to find this disease. However, since their work has been shown to work so many times before, it is more likely that our PCR cocktails did not go through enough troubleshooting to achieve success (Yaku et al. 2008). We even successfully ran Rosen's primers and got a band. Another implication of these results is that the site-directed mutagenesis did not provide a DNA template with the mutation. This would contradict previously published work on site-directed mutagenesis (Carter, 1986). Again, it is likely that more troubleshooting would lead to more information.

            The results of the sociological experiment clearly shows a correlation between having symptoms of this disease and feeling disabled, as well as having decreased physiological ability. This result supports our hypothesis and answers the question that ALS patients indeed feel more disabled over time. It also supports results obtained by a study on the quality of life of ALS patients (Foley et al, 2007). This study explained the benefits of coping mechanisms for such disabilities. Our results also support the results of a study done by the department of neurology in Minnesota (Mateen et al, 2008). This study attempted to use a mix of qualitative data and quantitative data to predict the duration of the disease. Our results show a significant increase in loss of physical activity over time, however our results alone are not enough to determine whether the trend is linear, exponential, or other.

Future Experimentation        

            This study has the ability to aid in the diagnosis of the A4V mutation on the SOD1 gene which causes ALS. Our failure does not completely reject the ability for our assay to work. More troubleshooting of the PCR cocktails may have led to success. Time-constraints led us to cease more attempts at optimizing the factors that go into PCR. One route of future study would be to attempt PCR with these primers using mutant DNA gathered from a research team, instead of being synthesized via site-directed mutagenesis.  Another possible study is to completely redesign the primers and attempt them with the successful site-directed primers. The sociological experiment helps future researchers have an idea of what the symptoms of ALS actually do to the patient sociologically and physiologically.  Since death is usually caused by respiratory failure, future research on the decline of breathing functionality is appropriate.

           The complexity of this experiment permits plenty of room for error, especially in regards to the primers used. One potential problem is that the primers need to anneal to the right spots on the DNA and the amplification process of PCR will not provide the targeted area as desired. If the forward primer were to anneal to the reverse primer, creating a primer-dimer situation, the PCR would not be conducted correctly again and the band on the gel would not be as anticipated (Brownie, et al, 1997). Another possibility is that the PCR primers do not have the correct annealing temperature to allow primers to anneal to the DNA. Each primer has an optimal temperature for which it will anneal to a DNA strand and is based upon various criteria like the amount of cytosine and guanine along with the concentrations of salts in the reaction (Don et al, 1991). One last obstacle for the annealing of the primers is the duration used at each of the temperatures. If the durations are not long enough, then the stages of PCR will not be correct and the entire process could be jeopardized (Yu and Pauls, 1992). If a mistake occurs in the first few cycles it could result in the band being in a different location on the gel than it should be (Yu and Pauls, 1992).